2-O-Sulfated Domains in Syndecan-1 Heparan Sulfate Inhibit Neutrophil Cathelicidin and Promote Staphylococcus aureus Corneal Infection

Abstract

Ablation of syndecan-1 in mice is a gain of function mutation that enables mice to significantly resist infection by several bacterial pathogens. Syndecan-1 shedding is induced by bacterial virulence factors, and inhibition of shedding attenuates bacterial virulence, whereas administration of purified syndecan-1 ectodomain enhances virulence, suggesting that bacteria subvert syndecan-1 ectodomains released by shedding for their pathogenesis. However, the pro-pathogenic functions of syndecan-1 ectodomain have yet to be clearly defined. Here, we examined how syndecan-1 ectodomain enhances Staphylococcus aureus virulence in injured mouse corneas. We found that syndecan-1 ectodomain promotes S. aureus corneal infection in an HS-dependent manner. Surprisingly, we found that this pro-pathogenic activity is dependent on 2-O-sulfated domains in HS, indicating that the effects of syndecan-1 ectodomain are structure-based. Our results also showed that purified syndecan-1 ectodomain and heparan compounds containing 2-O-sulfate motifs inhibit S. aureus killing by antimicrobial factors secreted by degranulated neutrophils, but does not affect intracellular phagocytic killing by neutrophils. Immunodepletion of antimicrobial factors with staphylocidal activities demonstrated that CRAMP, a cationic antimicrobial peptide, is primarily responsible for S. aureus killing among other factors secreted by degranulated neutrophils. Furthermore, we found that purified syndecan-1 ectodomain and heparan compounds containing 2-O-sulfate units potently and specifically inhibit S. aureus killing by synthetic CRAMP. These results provide compelling evidence that a specific subclass of sulfate groups, and not the overall charge of HS, permits syndecan-1 ectodomains to promote S. aureus corneal infection by inhibiting a key arm of neutrophil host defense.

Keywords: antimicrobial peptide (AMP); cathelicidin; heparan sulfate; host defense; keratitis; neutrophil; proteoglycan; syndecan.

FIGURE 1.

Syndecan-1 is the predominant HSPG in the mouse corneal epithelium and Sdc1^−/− mice specifically resist S. aureus corneal infection compared with Wt mice. A, eye sections (5 μm) of uninfected Wt mice were immunostained with 5 μg/ml 281-2 anti-mouse syndecan-1 ectodomain or 5 μg/ml Ky8.2 anti-mouse syndecan-4 ectodomain antibodies directly conjugated to Alexa 594 or 488 (original magnification, x200). B, scarified corneas of Wt, Sdc1^−/−, and Sdc4^−/− mice on the BL/6J background were infected topically with 5 × 10⁸ cfu of S. aureus strain 8325-4. The corneal bacterial burden was quantified 7 h after infection. Data shown are mean ± S.E. (n = 10 in Wt, n = 12 in Sdc1^−/−, and n = 14 in the Sdc4^−/− groups, *, p < 0.05 relative to Wt, Student's t test). C, scarified corneas of Wt and Sdc1^−/− mice on the BALB/c background were infected topically with 5 × 10⁸ cfu of S. aureus 8325-4, P1 or USA300, and the corneal bacterial burden was quantified at 7 h post-infection. Data shown are mean ± S.E. (8325–4: n = 8 for both Wt and Sdc1^−/−, P1: n = 14 for both Wt and Sdc1^−/−, and USA300: n = 14 for Wt and n = 12 for Sdc1^−/−; *, p < 0.05 relative corresponding Wt). D, eye sections (5 μm) of Wt and Sdc1^−/− mice isolated at 7 h after infection with S. aureus 8325–4 were Gram-stained (original magnification, ×200).

FIGURE 2.

Syndecan-1ectodomain HS specifically promotes S. aureus corneal infection in a dose-dependent manner. A, scarified corneas of Sdc1^−/− mice were infected topically with 5–6 × 10⁸ cfu of S. aureus 8325-4, administered vehicle (Sdc1^−/−, control) or 200 ng of purified syndecan-1 ectodomain, HS, CS, core protein (CP), or partially purified syndecan-4 ectodomain at 3 h after infection, and the corneal bacterial burden was quantified at 10 h after infection (n = 16 for the control group, n = 10 for the syndecan-1 ectodomain, HS and syndecan-4 ectodomain groups, n = 4 for the CS and CP groups; *, p < 0.05 relative to control). B, scarified Sdc1^−/− corneas were infected topically with 5 × 10⁸ cfu of S. aureus, administered increasing doses of purified syndecan-1 ectodomain or HS at 3 h post-infection, and the corneal bacterial burden was measured at 10 h post-infection (n = 4 for 20 ng, n = 6 for 85 ng, and n = 10 for 200 ng groups for both ectodomain and HS).

FIGURE 3.

Topical administration of 2-O-sulfated heparan compounds enhances S. aureus virulence in injured corneas. A, diagram of chemical structures of repeating disaccharide units of HS. B, chemical structures of repeating disaccharide units of heparosan and derivatives. C, scarified corneas of Sdc1^−/− mice were infected topically with 3 × 10⁸ cfu of S. aureus (Sdc1^−/−, control), administered 200 ng of heparin (HP), N-desulfated HP (NDS-HP), 2-O-desulfated HP (2ODS-HP), or 6-O-desulfated HP (6ODS-HP) at 3 h post-infection, and the corneal bacterial burden was quantified at 10 after infection (n = 6 in all groups; *, p < 0.05 relative to control). D, scarified Sdc1^−/− corneas were infected topically with 3 × 10⁸ cfu of S. aureus without (Sdc1^−/−, control), administered 500 ng of heparosan (H), N-sulfated H (NS-H), or N- and 2-O-sulfated H (NS2OS-H) at 3 h post-infection, and the corneal bacterial burden was measured at 10 h post-infection (n = 6 in all groups; *, p < 0.05 relative to control). The dose of HP and H compounds was based on preliminary titration experiments.

FIGURE 4.

2-O-sulfated domains in HS inhibit the staphylocidal activity of neutrophils. A, Wt neutrophils (5 × 10⁵) were incubated with 2 × 10³ cfu of pre-opsonized S. aureus in the absence (control) or presence of 3 μg/ml HP, NDS-HP, 2ODS-HP, or 6ODS-HP for 2 h at 37 °C. Percent bacterial killing was enumerated by plating serial dilutions of detergent lysates onto TSB agar plates (n = 4, *, p < 0.05 relative to control). B, Wt neutrophils (5 × 10⁵) were incubated with 2 × 10³ cfu of pre-opsonized S. aureus in the absence (control) or presence of 10 μg/ml H, NS-H, or NS2OS-H for 2 h at 37 °C and bacterial killing was determined (n = 8, *, p < 0.05 relative to control). The dose of HP and H compounds was selected on preliminary titration experiments with HP or NS2OS-H.

FIGURE 5.

2-O-sulfated domains in syndecan-1 HS inhibit extracellular killing mechanisms of neutrophils. A, Wt neutrophils (5 × 10⁵) were incubated with pre-opsonized S. aureus for 15, 30, 60, or 90 min in the absence or presence of 3 μg/ml HS, washed, incubated with 100 μg/ml gentamycin for 30 min to kill extracellular bacteria, washed, treated with TSB containing 0.1% (v/v) Triton X-100, and detergent lysates were plated out to determine the rate of phagocytic killing (mean ± S.E., n = 5). B, Wt neutrophils (5 × 10⁵) were pre-treated with 10 μg/ml cytochalasin D, incubated with S. aureus (2 × 10³ cfu) in the presence of cytochalasin D without or with 1 μg/ml purified ectodomain, 3 μg/ml HS or CS, or 10 μg/ml H or NS2OS-H for 2 h at 37 °C, and bacterial killing was determined (n = 22 for the cytochalasin D group, n = 11 for the +Ecto group, n = 16 for the +HS group, n = 4 for +CS and +H groups, and n = 5 for the +NS2OS-H group; *, p < 0.05 relative to the cytochalasin D group). C, supernatants from neutrophils stimulated with 1 μm fNLP for 1 h were incubated with S. aureus (1.5 × 10³ cfu) in the absence (fNLP sup) or presence of 1 μg/ml ectodomain, 3 μg/ml HS or CS, or 10 μg/ml H or NS2OS-H for 2 h at 37 °C, and bacterial killing was determined (n = 5 in all groups; *, p < 0.05 relative to the fNLP sup group).

FIGURE 6.

Neutrophil extracellular killing of S. aureus is mediated by CRAMP, and syndecan-1 ectodomain and 2-O-sulfated heparan compounds inhibit killing of S. aureus by CRAMP. A, supernatants from Wt neutrophils stimulated with fNLP were incubated with S. aureus (1.5 × 10³ cfu) or were immunoprecipitated with mouse IgG, anti-CRAMP, anti-MPO, or anti-lactoferrin antibodies and then incubated with S. aureus for 2 h at 37 °C, and % bacterial killing was determined (n = 5 in all groups, *, p < 0.05 compared with the fNLP sup group). B, CRAMP (10 μg/ml) was pre-incubated with vehicle or 10 μg/ml purified ectodomain, HS, H, or NS2OS-H for 15 min and then incubated with S. aureus (10³ cfu) for 2 h at 37 °C, and % bacterial killing was determined (n = 6 in all groups; *, p < 0.05 compared with the CRAMP only group). C, S. aureus incubated with vehicle, CRAMP, or CRAMP and ectodomain, HS, H, or NS2OS-H were stained for live and dead bacteria.